Cellular cholesterol requirements are regulated through a negative feedback loop that responds to elevations in intracellular cholesterol. Central to this pathway are sterol regulatory element-binding proteins (SREBPs) that activate expression of genes involved in the synthesis and uptake of cholesterol. In the ER, cholesterol modulates SREBP processing through its binding to the SREBP cleavage activating protein (SCAP) and induction of conformational change. The regulatory ER cholesterol pool appears to be plasma membrane (PM)-derived, though the mechanism by which PM cholesterol is transferred to ER membranes is poorly understood. Biochemical and genetic studies suggest that PM lipids play a critical role in movement of PM cholesterol to the ER. Our central hypothesis is that the mechanism(s) governing the transfer of PM cholesterol to the ER membranes critically depend upon the organization and composition of PM lipids and PM structure. We propose that a possible route of cholesterol transfer is via direct PM-ER membrane contacts. Oxysterols, which are physiological regulators of sterol homeostasis, may modulate PM to ER cholesterol transfer through direct interaction with PM lipids, leading to displacement of PM cholesterol for transfer to ER membranes. Alternatively, oxysterols may facilitate transfer of PM cholesterol to the ER through direct interaction with ER membranes by promoting ER-PM fusion. The goals of this project are to identify the machinery involved in this sterol trafficking pathway and to investigate the molecular mechanisms involved in cholesterol transfer between PM and ER. We will test our central hypothesis by 1) using a genetic screen to isolate Chinese hamster ovary (CHO) cell mutants with cholesterol trafficking defects, 2) characterization of the effect of the genetic defects in the CHO mutants on PM lipid composition and morphology, and on PM to ER cholesterol trafficking, and 3) performing biophysical studies with unique cholesterol and oxysterol probes to examine the mechanism of PM cholesterol transfer to ER membranes. Studies outlined in this proposal will contribute to our understanding of the mechanisms involved in regulation of ER cholesterol homeostasis, and may identify strategies for protection from the cytotoxic effects of cholesterol overload.